Sequence Analysis of Different Domains of Plasmodium vivax Apical Membrane Antigen (PvAMA-1 gene) Locus in Iran.

BACKGROUND
Plasmodium vivax is responsible for approximately 80 million malaria cases in the world. Apical membrane antigen1 (AMA-1) is a type I integral membrane protein present in all Plasmodium species. AMA-1 interferes in critical steps of invasion of human hepatocytes by sporozoites and red blood cells by merozoites and is one of the most immunodominant antigens for eliciting a protective immune response in human. It is considered as a promising antigen for inclusion in a vaccine against P. vivax. Since more knowledge is needed to lighten the scope of such antigen we compared genetic variation in P. vivax AMA-1from an Iranian isolate with those reported from some of the other malarious countries so far.


METHODS
P. vivax genomic DNA was extracted from the whole blood of an Iranian patient with patent P. vivax infection. The nucleotide sequence for 446 amino acid (AA) residues (42-488 of PvAMA-1) was amplified by PCR and cloned in pUC19 vector for sequencing.


RESULTS
Sequence analysis of the antigen showed a high degree of identity (99%) with strong homology to the PvAMA-1 gene of P. vivax S3 and SKO814 isolates from India and Korea (Asian isolates) respectively, and 96% similarity with P. vivax Sal-1 AMA-1 gene from El Salvador.


CONCLUSIONS
We cloned and characterized three domains of PvAMA-1 gene from an Iranian patient. Predicted protein sequence of this gene showed some discrepancies in corresponding protein in comparing with similar genes reported from other malarious countries.


Introduction
ne of the most widespread human malaria parasites is Plasmodium vivax and about 2.6 billion people are at risk of the infection in the world (1). The infection causes approximately 132-391 million episodes of disease each year (2). Although P. vivax is the main responsible for malaria morbidity outside Sub-Saharan Africa, it has received little attention and limited funds for research and control owing to producing less severity than P. falciparum (3). Moreover, non-cultivable nature of P. vivax has induced some limitations for the study of its molecular biology up to now. Indeed, increasing the knowledge of genetic diversity and population structure of this parasite will result in the development of an effective vaccine to control the disease (4). Apical membrane antigen (AMA-1) is a member of a group of molecules which are quite conserved in Plasmodium species (5) and are expressed on the micronemes and surface of merozoite in all Plasmodium species (6). AMA-1 is expressed in the late schizont stage of the asexual plasmodia life cycle (7). The antigen plays a unique role in critical stage of invasion the human hepatocytes by sporozoites (8) and red blood cells by merozoites (9)(10)(11)(12). This gene shows few predominant haplotype and displays very limited genetic diversity within any geographic region so more investigations are needed to confirm these observations (13). Moreover, AMA-1 is one of the most immunodominant antigens which are considered as promising antigen for the development of a recombinant vaccine against P. vivax (7,14,15). It could be also used for immunological studies to evaluate the natural acquired antibody response to P. vivax AMA-1 (16). Therefore, polymorphism investigation of this gene in different areas could result in developing diagnostic kits.
Iran is one of the endemic areas for P. vivax. The parasite possesses the most reported cases of the malaria parasites each year in the country (17). So it is important to investigate the molecular nature of the parasite nested in Iran in comparing with the same species of the parasites in the other malarious areas. Because of strong candidate of PvAMA-1 for vaccine development and producing diagnostic kits, the PvAMA-1 was sequenced and then compared with similar genes recorded in GenBank.

Parasite sample
The case was an Iranian inhabitant of Jask district (Hormozgan Province) with symptomatic P. vivax infection. Blood of the patient was collected into the EDTA tube using venipuncture method. P. vivax infection was documented by microscopic analysis using Giemsa stained thick and thin blood smears.

DNA extraction
The genomic DNA was extracted from the whole blood using Genomic DNA Extraction Kit, ACCUPreP® kit (BIONEER) according to the manufacturer's instructions. The quality and quantity of the extracted DNA was checked using a biophotometer (Ependorf) at 260 and 280 nm and electrophoresis on 1% agarose gel.

Results
As Fig. 1 shows a 1300bp band has been resulted from extracted genomic DNA by PCR. The production of PCR was cloned in pUC19 and then sequenced. The sequenced gene was submitted to the GenBank and registered under the HM535663.1 accession number. The predicted protein sequence analysis revealed that it composes of 446 amino acids including 3 domains and sixteen Cysteine residues (Fig. 2).
In conclusion, at the present study, we cloned and characterized three domains of PvAMA-1 gene from an Iranian patient. Predicted protein sequence of this gene obviously has a high degree of identity (99%) with strong homology to the PvAMA-1 gene of P. vivax with Asian isolates and also has 96% similarity with PvAMA-1 gene from El Salvador isolate.